Age-related lipotoxicity is defined as the accrual of toxic lipid intermediates such as fatty acyl CoA, ceramide, and diacylglycerol which act to inhibit aspects of tissue function. These metabolites accrue due to increased fatty acid biosynthesis, decreased fatty acid oxidation, and/or increased biosynthesis of the specific intermediate or decreased clearance of the specific intermediate. Aging skeleton has been found to accumulate greater amounts of lipid at the expense of mineralizing bone. Published data as well as our preliminary data indicated BMSC osteoblastic differentiation in vitro and new bone formation in vivo are resistant to stimulatory effects of BMP in older mice. Additionally, we have found that BMP-7-induced ectopic bone in old mice contained elevated levels of FAS compared to young mice. Moreover, we observed that the mTOR inhibitor rapamycin (RAPA) inhibits BMP-7-induced osteogenesis and lipogenesis in cultured osteoblast cells from young animals. We hypothesize that toxic lipids accrue in aged bone and that blockade in the accrual of these lipids will restore BMP-7-induced osteoblast differentiation. We also hypothesize that mTOR determines the balance between osteogenesis and lipid accrual in bone cells, and that this balance may shift towards lipid accrual at the expense of bone formation in aging bone. To test these hypotheses, we will first utilize in vitro cultures of osteoprogenitor cells from bone marrow and calvariae of young, middle aged, and old mice to determine whether lipid intermediates accrue to a greater degree in cells from the older animals and to determine whether pharmacologic and molecular genetic interventions designed to directly decrease lipid accrual and to inhibit mTOR will restore basal and BMP-7-induced osteoblast differentiation. In Aim 2, we will utilize an in vivo model of BMP-7-induced ectopic new bone formation to determine whether direct inhibition of lipid accrual will restore the ability of BMP-7 to promote optimal new bone formation in aging mice. Outcome of this work will provide heretofore unavailable data that will illustrate mechanisms of age-related skeletal lipotoxicity, and whether maneuvers which block toxic lipid accrual can restore bone formation. These outcomes will provide the necessary basis for the development of future therapeutics for the treatment of osteoporosis aimed at reducing lipotoxicity. PUBLIC HEALTH RELEVANCE: Osteoporosis is a serious disease in the aging population characterized by bone weakening as the result of insufficient bone formation rates, which has recently been attributed to fat accumulating in aging bone. This project will determine the causes and consequences of toxic lipid accrual in bone, whether the rapamycin-inhibitable mTOR pathway exerts antagonistic effects to increase both formation of mineralized bone and fat in the skeleton, and whether the fat forming component of this pathway can be blocked in order to preserve and optimize its bone forming action. Successful outcomes will pave the way for practical therapies designed to minimize the fat forming action of the mTOR pathway and maximize its bone forming actions.